We have been studying assembly of adenovirus with attention drawn to the polar encapsidation of the viral genome in vivo and the interactions of empty viral capsids with DNA molecules in vitro. Molecular cloning, sequence determination, and analysis of novel genome variants of adenovirus type 3 (Ad3) have delimited the left end DNA sequence recognized for polar encapsidation. Host range variants have enabled us to discern and exploit interference and cross-subgroup complementation in the expression of early region Ia, part of the adenovirus transformation/tumor antigen complex. We have further characterized the avid binding of empty adenovirus capsids with DNA in vitro, in particular with closed circular, superhelical DNA substrates. Most recently we have successfully used the capsid:DNA complexes as an efficient gene transfection system, demonstrating adsorption to a variety of cells in culture, expression of transfected genes, infectivity of transfected papovavirus DNA, and cellular transformation by a cloned thymidine kinase gene. One objective now proposed is to establish and transpose the DNA encapsidation sequence and evaluate the capacity for cross subgroup recognition of this site by the presumed viral proteins that play a role in DNA packaging. As part of this effort we want to evaluate the evolutionary constraints and divergence of the encapsidation site and the proximal (perhaps overlapping) sequences that regulate expression of early gene Ia. Methods of site directed mutagenesis and gene transfer that have been developed, some by this laboratory, will be used in this proposed investigation of the structure, functions and evolution of non-coding sequences near the left end of the adenovirus genome. This will be of immediate interest in terms of adenovirus biology, but also for the broader fields of gene expression and genetic modification of animal cells.